Cell migration occurs in all multicellular organisms. Because cell migration can be beneficial to an organism, as in development, or detrimental, as in cancer, it is important to understand the nuances of this process both at the cellular and molecular level. The goal of this proposal is to determine the role of the transcription factor, Fd64a, in Drosophila embryonic development. Fd64a is a member of the Fox family of transcription factors, which have multiple different roles, including modulating the expression of genes involved in migration. fd64a is expressed in a subset of Drosophila muscles that directly contact the migrating salivary gland, an ideal and unique model system for studying cell migration in the context of an organ. In flies deficient for fd64a, the salivary gland exhibits a range of migration defects. These preliminary findings led to the hypothesis that Fd64a regulates expression of molecules that direct salivary gland migration. To further investigate the role of fd64a in cell migration, Part A of Specific Aim 1 will be to create a knock-out of fd64a by homologous recombination, and then analyzing this null allele for salivary gland migration and musculature defects. Part B of Specific Aim 1 will test if over-expression of fd64a can affect salivary gland migration. Additionally, this aim will determine if the knock-out defects can be rescued by providing wild-type fd64a function in a tissue-specific manner. Because fd64a is expressed in multiple different tissues of the Drosophila embryo, the goals of Specific Aim 2 will be to first positively identify the tissues that express fd64a and then to examine the development and morphology of these tissues in the fd64a null allele. Specific Aim 3 will investigate the downstream targets of Fd64a. Part A will use a candidate gene approach. Expression of Semaphorin 2A, a molecule known to affect migration of multiple other cell types, overlaps that of fd64a, suggesting a potential regulatory interaction. I will use in situ hybridization, protein expression, and phenotypic analysis to determine if semaphorin 2A is a viable target of Fd64a. Part B will identify other targets of Fd64a via microarray analysis comparing changes of gene expression in wild-type and fd64a-null embryos. Potential targets will be verified by in situ analysis, chromatin IP, and mutant-allele phenotype analysis. The molecular and genetic analysis of fd64a in the Drosophila embryo will provide insight into how cues provided by surrounding tissues influence migration of specific organs. This represents an important contribution to the broadening field of cell migration.